Milk is a complex fluid capable of sustaining the total nutrition of the human infant for 6 months or longer. To meet the caloric and nutritional demands of newborns, mammary epithelial cells synthesize and secrete large quantifies of triglyceride (TAG) into milk during lactation. Milk lipids originate from protein-coated triglyceride droplets (CLD), which are synthesized at the endoplasmic reticulum (ER) and transported to the apical plasma membrane where they are secreted by a unique membrane envelopment process. Several lines of evidence indicate that CLD production by milk secreting cells is developmentally regulated, and that it is induced during differentiation of the mammary gland into a secretory organ by sequential activation of processes that inhibit TAG lipolysis and stimulate TAG synthesis. Disruption of CLD induction has significant physiological consequences including impaired glandular development and lactation failure. The long-term objectives of our laboratory are to elucidate the molecular and cellular mechanisms underlying regulation CLD development in differentiating milk secreting cells, and how impairment of CLD development interferes with mammary gland differentiation. Our studies indicate that TAG lipolysis is mediated by adipose triglyceride lipase (ATGL), and that inhibition of ATGL lipolysis by the CLD coat protein adipophilin (ADPH) is responsible for initiating CLD production during the initial phase of mammary gland differentiation. Subsequent CLD accumulation is hypothesized to depend on AKTI dependent activation of TAG synthesis through a mechanism that involves modulation of the activity of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1), the enzyme responsible for synthesizing TAG. We propose to test these hypotheses by determining the specific contributions of ADPH inhibition of TAG lipolysis and DGAT1 dependent TAG synthesis to CLD development in the differentiating mouse mammary gland, and to define the specific physiological roles of AKTI activity in regulating this process. Because lipid synthesis in the mammary gland is robust and developmentally regulated by well defined promoter systems, and mammary epithelial cells can be manipulated by transgenic and adenoviral techniques, the proposed studies offer an excellent opportunity to understand molecular interactions involved in TAG synthesis and storage that may have applicability in other cells and tissues. RELEVANCE (See instructions): Milk lipids, mainly triglcyerides, supply the majority of the calories for neonatal growth and the essential fatty acids needed for neonatal membrane synthesis and the synthesis of bioactive lipid signalling molecules. Disrupting mammary gland triglyceride metabolism impairs milk lipid precursor formation and inhibits glandular differentiation producing lactation failure. This proposal focuses on understanding how milk lipid formation is regulated and the role of this process in mammary aland development.